Project Summary/Abstract Gut-microbiome can produce a variety of micronutrients that are important to human health in myriad aspects of metabolism. Queuine is one of the most intriguing and enigmatic of microbiome/diet-derived micronutrients and it is synthesized exclusively by eubacteria and salvaged by eukaryotes. Queuine can be actively taken up by all mammalian cells to be incorporated into the wobble anticodon position of four transfer RNAs (Tyr/His/Asn/Asp). Q-tRNA modification levels are highly dynamic and reflect the interplay between host cells and its gut microbiome. The queuosine modification-mediated regulatory role in cells was considered to significantly affect translation fidelity and translation efficiency through codon-biased protein synthesis for specific mRNA transcripts. Gut microbiota represents a complex ecosystem that develops in close parallel with hosts. Perturbations of profile and function of gut microbiome (dysbiosis) can lead to altered host responses that increase the risk of diseases. The association of gut-microbiome derived Q-tRNA with digestive diseases and the molecular mechanism by which Q-tRNA modifications modulate the disease status remain to be investigated. This proposal aims to investigate molecular and cellular mechanisms of Q-tRNA modification and microbiome-dependent phenotypes, using cultured intestine cells and mouse models with dysbiosis. The developed method to detect and quantify Q-tRNA modification will be used to test the response of human intestine cells to micronutrient queuine, cells with significant cellular phenotypes will be prepared for comparative mRNA-sequencing and ribosome profiling in combination with proteomic analysis. To further investigate the physiological role of Q-tRNA modifications in vivo, inflammatory bowel disease (IBD) mice model treated with queuine will be established. Physiological phenotypes and microbial species will be examined, and Q-tRNA modification levels from different tissues will be measured to correlate with mRNA-Seq and proteomic results. Potential Q-tRNA-mediated gene targets will be validated in mouse with inflammatory bowel disease. This proposal is important to understand the host-microbiome interactions through Q-tRNA modification and the expected results will provide new insights into molecular mechanisms underlying microbiome-mediated digestive diseases. The state-of-the-art technologies applied in this proposal will lay the groundwork for the study of epitranscriptome in gut-microbiome mediated intestinal inflammation. During this project, I will make use of my expertise in biochemistry and medicine, and work closely with my mentors and collaborators to extend my knowledge and experimental skills in biochemistry, cell biology, RNA sequencing and mouse work to address key questions related to Q-tRNA modification. This K01 award will not only allow me to study the above fundamental questions, but also will prepare me for an independent career in research in the future.